The field of the invention relates to compounds that inhibit the biological activity of nitric oxide synthases (NOSs). In particular, the field of the of the invention relates to compounds that selectively inhibit nitric oxide synthases such as human neuronal NOS (nNOS) and bacterial NOS (bNOS).
NO is a highly reactive free radical produced by the hemethiolate monooxygenase nitric oxide synthase (NOS, mNOS=mammalian NOS, bNOS=bacterial NOS). NOS generates NO by oxidizing L-Arg and is found in both mammals and some bacteria. While mNOS is a multi-domain protein composed of both oxygenase and reductase domains, bNOS from the genus Bacillus and Staphylococcus contains only an oxygenase domain. X-ray crystal structures determined for both bNOS and mNOS oxygenase domains reveals a near identical tertiary structure and active site except that bNOS lacks the N-terminal fragment that contains the Zn2+ binding motif observed in mNOS.
In mammalian systems, NO functions as an essential signaling molecule and is involved in a variety of physiological functions ranging from blood pressure homeostasis to neural cell communication and host defense. There are three mNOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Owing to the pathological consequences of the over or under production of NO, significant effort has been made toward the development and characterization of isoform selective mNOS inhibitors, which has resulted in the development of many unique inhibitors.
One of the major issues in the design of bNOS inhibitors is its structural similarity to mNOS isoforms. Direct comparison of the mammalian and bacterial NOS structures/sequences reveals several key differences that could be exploited for a bNOS inhibitor design effort. The first key difference is between the domain architecture of the NOS isoforms. Each mNOS is a multi-domained protein composed of both a reductase and oxygenase domain whose activity is regulated by calmodulin. In sharp contrast, bNOS is only composed of an oxygenase domain and is not regulated by calmodulin. Since bNOS is not covalently linked to its redox partners like mNOS, bNOS must utilize redox partners for activity. A second key difference is amino acid variances between the NOS active sites. For example, both bNOS and endothelial NOS (eNOS) have an Asn residue that directly interacts with the L-Arg substrate while this residue is Asp in nNOS and inducible NOS (iNOS). The active site Asp/Asn difference provided the initial structural underpinning for the design of nNOS selective inhibitors. Despite this difference in electrostatics between bNOS and nNOS, inhibitors that target the Asn residue might be detrimental if they also inhibit the critical eNOS isoform. Additional active site differences in bNOS include His128 (mammalian equivalent is Ser) and Ile218 (mammalian equivalent is Val). The slightly bulkier Ile adjacent to the O2 binding site has been shown to decrease the NO release rates in bNOS. The last key difference between mNOS and bNOS is present at the pterin cofactor-binding site. Since bNOS lacks the N-terminal Zn2+ binding motif present in mNOS, the pterin binding site is more exposed in bNOS, resulting in weaker micromolar binding affinity in bNOS vs. the stronger nanomolar affinity in mNOS. While the physiologically relevant bNOS cofactor that binds to the bNOS pterin site remains unknown, inhibitors that target this site are an attractive avenue for structure-based drug design.
In Gram-positive bacteria, bNOS produced NO has been found to modulate macromolecules by nitrosylation, to function as a commensal molecule, to protect against oxidative stress, and to detoxify antimicrobials (See, e.g., Gusarov, I. and Nudler, E. NO-mediated cytoprotection: instant adaption to oxidative stress in bacteria. Proc. Natl. Acad. Sci. USA 102, 13855-13860 (2005); and Gusarov, I., Shatalin, K., Starodubtseva, M. and Nudler, E. Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. Science 325, 1380-1384 (2009)). Although the biological function of NO varies among bacterial organisms, the unique ability of NO to protect the pathogens Staphylococcus aureus and Bacillus anthracis against oxidative and antibiotic-induced oxidative stress by activation of catalase and by suppression of damaging Fenton chemistry implicates bNOS as a potential therapeutic target (Gusarov, supra). Moreover, commonly used antibiotics for the treatment of Gram-positive pathogens-like beta-lactams and vancomycin-elicit antibacterial function by generation of reactive oxygen species. Together, these data suggest that inhibition of bNOS will attenuate bacterial survival against antibiotic induced oxidative stress. Owing to the essential role NO plays in mammals, development of a bNOS-specific inhibitor ideally should take advantage of subtle differences between bNOS and mNOS.
To do so first requires identification of NOS inhibitors that demonstrate antimicrobial-like properties within a bacterial system under oxidative stress and characterization of the inhibitor-binding mode for structure-based inhibitor development. Studies on the effects of inhibitors on bNOS have thus far been limited to the finding that nonselective NOS inhibitor NG-methyl-L-arginine generates greater sensitivity to H2O2-induced oxidative stress in B. anthracis. Accordingly, there is an ongoing search in the art for NOS inhibitors that decrease bacterial viability in the presence of an antimicrobial agent or otherwise under conditions inducing oxidative stress.
As bacterial pathogens acquire resistance to commonly used antibiotics, it has become clear that novel therapeutic strategies are required to combat serious infections. In particular, there is an urgent need for the development of new pharmaceuticals that target methicillin-resistant Staphylococcus aureus (MRSA). MRSA, a Gram-positive pathogen resistant to common antibiotics like isoxazoyl penicillins and β-lactams, was first reported in 1961 and remains one of the most costly bacterial infections worldwide. MRSA has remained a major threat to public health in part due to the emergence of community-associated strains, its varying epidemiology, and drug resistance. In recent years, the threat of MRSA has been compounded by reports of vancomycin resistant strains, as this agent is often considered the drug of last resort. Therefore, characterization and exploitation of alternative bacterial drug targets is essential for future successful management of MRSA infections.
Recent gene deletion experiments in S. aureus, B. anthracis and B. subtilis have implicated bacterial nitric oxide synthase (bNOS) as a potential drug target, as it provides the bacterial cell a protective defense mechanism against oxidative stress and select antibiotics. The growth of B. subtilis was found to be severely perturbed in response to combination therapy with an active site NOS inhibitor and an established antimicrobial.
While such evidence suggests bNOS as a potential therapeutic target for improving the efficacy of antimicrobials, design and development of a potent bNOS inhibitor is complicated by the active site structural homology shared with the three mammalian NOS (mNOS) isoforms. Especially considering the critical role of mammalian iNOS in pathogen clearance, bNOS inhibitors must be isoform specific to circumvent short-circuiting critical mammalian NO functions. Recent structure-based studies suggest that bNOS specificity can be achieved through targeting the pterin-binding site, as the bNOS and mNOS pterin binding sites are quite different.